Familial hypercholesterolemia (FH) is caused by mutations in various genes, including the
Familial hypercholesterolemia (FH) inheritable disorder of abnormal low-density lipoprotein (LDL) metabolism that is characterized by elevated plasma concentrations of total cholesterol (TC) and LDL cholesterol (LDL-C), xanthomas (cholesterol deposits in the skin and tendons) and an increased risk of premature coronary artery disease (CAD). Homozygous FH affects 1 in 1,000,000 individuals, and the frequency of heterozygous FH varies from 1 in 200 to 1 in 500 individuals, depending on the population (
Monogenic FH is caused by defects in several genes that encode proteins involved in LDL uptake and catabolism (
Hypercholesterolemia may be associated with other rare disorders of lipid metabolism, which have very similar clinical presentations; one example is sitosterolemia, which is caused by mutations in genes encoding either of two ATP-binding cassette (ABC) transporters, ABCG5 or ABCG8, which limits intestinal absorption and promotes biliary excretion of sterols (
Despite being one of the most common genetic disorders, FH still remains largely undetected and untreated worldwide (
The spectrum and prevalence of FH-related mutations remains to be studied in Russia. Previous studies have used limited genetic screening methods based on PCR and Sanger sequencing to study specific relevant genes, such as
The present study was approved by the Ethics Committees of Center for Atherosclerosis and Lipid Disorders of North-Western District Scientific and Clinical Center Named After L.G. Sokolov, Medical Faculty of Saint-Petersburg State University, City Hospital No. 40 (St. Petersburg, Russia) and Research Centre for Medical Genetics (Moscow, Russia), where patients were treated, and genetic analysis was performed. Written informed consent was obtained from all patients or the children's legal representatives prior to the beginning of the study.
A total of 59 unrelated citizens from Saint-Petersburg and Moscow (29 male/30 female) with suspected FH were enrolled in the present study. Clinical data were collected, including the prior lipid levels, family and personal history of dyslipidemia and the presence of premature atherosclerotic cardiovascular disease (ASCVD), as well as the presence of tendon/skin xanthomas and lipoic corneal arcus. Demographic characteristics and clinical features of the groups are presented in
The adult group included 31 patients (12 males and 19 females; median age, 49; age range, 23-70) who fulfilled the Simon Broome criteria for definite/possible FH (
NGS was performed as a collaboration between two genetic laboratories from Saint-Petersburg and Moscow, and the Illumina MiSeq (Illumina, Inc.) and Ion S5 (Thermo Fisher Scientific, Inc.) sequencing systems were used, respectively. Genomic DNA (gDNA) was extracted from whole blood using the Magna Pure system (Roche Diagnostics) or with the use of a Diatom DNA Prep reagent kit (Biocom) according to the manufacturer's protocols. Concentration of gDNA as well as DNA concentration of the libraries afterwards was determined using a Quantus Fluorometer™ (Promega Corporation) or Qubit™ Fluorometer (Thermo Fisher Scientific, Inc.). gDNA was subjected to electrophoresis in 1% agarose gel and the optical density ratio was used to confirm its integrity and purity.
DNA samples were prepared for the targeted NGS covering all of the coding exons of the
For sequencing on the Illumina platform, DNA libraries were prepared from 200 ng using a KAPA LTP Library Preparation kit with a custom designed SeqCap® EZ Choice Library Enrichment kit [Roche Diagnostics; cat. no. KK8232 (07961880001) and 170911_HG19_gb40_cardio_EZ_HX3, respectively]. Validation of the libraries was performed on the Agilent 4200 Tape Station (Agilent Technologies, Inc.). Concentration in nmol was calculated based on the size of the libraries and concentration in ng/µl. Libraries were normalized to 4 nmol before pooling and denaturation to get a final loading concentration of 12.5 pmol. Paired-end sequencing of the 150 bp libraries was performed on an Illumina MiSeq Sequencer (Illumina, Inc.) using a MiSeq Reagent kit v2 (300 cycles) (Illumina, Inc., cat. MS-102-2002) to obtain the FastQ data.
For sequencing on the Ion S5 system, DNA libraries were constructed with the Ion AmpliSeq™ custom panel and Ion AmpliSeq™ Library kit 2.0 (Thermo Fisher Scientific, Inc.; cat. nos. 04779971_Dyslipidemia_IAD175748_182 and 4480442, respectively). DNA quality was confirmed by the final stage of library preparation using test PCR performed with the included manufacturer's primers to adaptors sequences. The thermocycling conditions used were as follows: 95˚C for 40 sec, 68˚C for 35 sec and 72˚C for 75 sec; the number of cycles used was dependent on the library concentration. PCR results were visualized on silver-stained 8% acrylamide gel (staining time 10 min at 4˚C). Massive parallel sequencing of pooled libraries with loading concentration of 75 pmol was performed using an Ion 540™ Chip kit (Thermo Fisher Scientific, Inc., cat. A27766) and Ion 540™ Kit-Chef (Thermo Fisher Scientific, Inc., cat. mo. A30011) with an average amplicon length of 175 bps.
The 1000 Genomes human reference genome assembly (b37) was used for data analysis (
Variant validation was performed by PCR-direct sequencing. Specific primers were designed for verification in each case (
The NGS-based technique allowed for the identification of 32 different pathogenic/likely pathogenic variants, 7 of which had not been previously reported, in 43 patients (
In the adult group, pathogenic/likely pathogenic variants were detected in 18 (58%) patients: 13 Patients had FH-causing mutations in the
In total, 23 mutations were found in the
Novel frame-shift variants in the
A novel variant in exon 8, c.1186G>C p.(Gly396Arg), was found in a young man (aged 29) who had no family history of either hyperlipidemia or ASCVD. The patient had a maximal TC level of 9.7 mmol/l. During the period of genetic testing, the patient underwent a coronarography that showed preclinical diffuse atherosclerosis (30%) of the anterior interventricular artery. The p.(Gly396Arg) variant was predicted by
Another three new missense variants encoded in the ligand-binding domain of LDLR, as well as one splicing variant, were predicted by software tools as disease causing. c.325T>G p.(Cys109Gly), c.401G>C p.(Cys134Ser) and c.616A>C, corresponding to a known protein substitution c.618T>G p.(Ser206Arg), were detected in children from families with a history of hypercholesterolemia. These three patients, aged 11, 7 and 6 years old, respectively, demonstrated the highest LDL-C levels in the children/adolescent group. A splicing variant in intron 6 c.940+1_c.940+4delGTGA was detected in four unrelated individuals. This variant alters a canonical splice donor site. Previously, a similar splicing mutation, which affects 15 nucleotides (c.940_940+14del15), was described in a 21-year-old Spanish man who suffered a premature MI at the age of 16 years (
Previously, NGS has been successfully used for mutation screening of the
According to a previous publication, the mutation detection rates in patients with suspected FH varies from 20-90%, depending how rigorous the criteria used for selection of patients were (
The present study identified several novel
Identified variants were located mostly in coding regions which correspond to functional protein domains or produce truncated proteins. These variants do not overlap with any genomic regulatory regions. According to ENCODE in the UCSC Genome Browser, Tracks H3K27Ac, DNase Clusters and Txn Factor ChIP (data not shown), there was a highly probable presence of regulatory sequences in exon 1, intron 1-2, 3'-untranslated region (UTR) of the
A proband with a family history of high TC levels was homozygous for a c.894G>A splicing mutation in the
To the best of our knowledge, the present study is the first to identify mutations in the
As
A known
As mutations in the
In total, 16
A further 13
The present study has some limitations. For example, analysis of introns/3'UTRs of the studied genes was not performed, and co-segregation analysis could not be performed due to an insufficient number of patients for linkage disequilibrium studies.
In conclusion, the mutation spectrum for FH in Russian individuals is similar to that of other European countries. Evidence of this conclusion is that certain
Not applicable.
This work was partly supported by the Russian Science Foundation (grant no. 14-50-00069) and the state assignment of Ministry of science and Higher Education of the Russian Federation for RCMG.
The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions. All novel variants have been submitted to the ClinVar database (
EYZ, OSG, ASG, AMS, SNP designed and conceived the methodology of the present study, organized experiments and performed final interpretation of the data. MVM, SAU, VSG, SPU, SGS, IVA and DMG collected the patient data and assisted with clinical data interpretation. OVR, ONI, NAS, MAF and VVM performed the experiments. YAB, AAP, VVM, MAF and ONI performed the bioinformatics analysis and variant annotation. VVM wrote the first draft of the manuscript. All authors read and approved the final manuscript.
This study was approved by the Ethics Committees of Center for Atherosclerosis and Lipid Disorders of North-Western District Scientific and Clinical Center Named After L.G. Sokolov, Medical Faculty of Saint-Petersburg State University, City Hospital No. 40 (St. Petersburg, Russia) and Research Centre for Medical Genetics (Moscow, Russia) where patients were treated and genetic analysis was performed. Written informed consent was obtained from all patients or their legal representatives before the study.
Not applicable.
The authors declare that they have no competing interests.
Sanger sequencing results for next-generation sequencing-determined novel
Clinicopathological and demographic characteristics of the recruited cohort.
Characteristic | Adult, n=31 | Children/adolescent, n=28 |
---|---|---|
Age, years |
47.4±14.1 | 11.0±5.1 |
Age range, years | 23-70 | 2-21 |
Male, n (%) | 12(39) | 17(61) |
Female, n (%) | 19(61) | 11(39) |
Family history, n (%) | 23(74) | 24(86) |
Maximal total cholesterol, mmol/l |
11.0±2.2 | 9.3±1.4 |
LDL cholesterol, mmol/l |
6.8±2.5 | 6.7±1.8 |
Tendon xanthomas, n (%) | 22(71) | 0 (0) |
Lipoic corneal arcus, n (%) | 2(6) | 0 (0) |
Clinical and instrumental manifestations of ASCVD, n (%) | 17(55) | 0 (0) |
Increased intima-media thickness without clinical symptoms, n (%) | 3(10) | 0 (0) |
Patients on lipid-lowering therapy, n (%) | 31(100) | 1(4) |
aMean ± standard deviation. ASCVD, atherosclerotic cardiovascular disease; LDL, low density lipoprotein.
Characterization of genetic variants identified in the present study and their pathogenicity analysis.
A, Novel |
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Patient ID | Genetic variant | Number of patients | Variant ID in dbSNP or alternative database | Genomic position (GCRh37/hg19) | Pathogenicity analysis |
Functional domain | Pathogenicity |
SIFT | Mutation taster | Poly Phen2 | (Refs.) |
1 | Missense Exon 4 c.325T>G p.(Cys109Gly) | 1 | 869387 in ClinVar | Chr19: 11215907 | - | Ligand-binding | Likely pathogenic (PS1 PM1 PM2 PM5 PP3) | D | D | D | - |
2 | Missense Exon 4 c.401G>C p.(Cys134Ser) | 1 | 869388 in ClinVar | Chr19: 11215983 | - | Ligand-binding | Likely pathogenic (PS1 PM1 PM2 PM5 PP3) | D | D | D | - |
3 | Frameshift Exon 4 c.433_434dupG p.(Val145Glyfs*35) | 1 | 870329 in ClinVar | Chr19: 11216013 | - | Ligand-binding | Pathogenic (PVS1 PM2 PP3) | D | D | -- | - |
4 | Missense Exon 4 c.616A>C p.(Ser206Arg) | 1 | 869389 in ClinVar | Chr19: 11216198 | - | Ligand-binding | Uncertain value (PM2 PP3) | D | D | D | - |
5 | c.940+1_c.940+ 4 delGTGA (g.18154_18157 delGTGA) | 4 | 869390 in ClinVar | Chr19: 11218191-11218194 | - | Splice donor site, intron 6 | Pathogenic (PVS1 PM1 PM2 PP3) | -- | D | -- | - |
6 | Missense Exon 8 c.1186G>C p.(Gly396Arg) | 1 | 870321 in ClinVar | Chr19: 11222315 | - | EGF precursor homology B repeat | Pathogenic (PVS1 PM1 PM2 PM5 PP3) | D | D | D | - |
7 | Frameshift Exon 11 c.1684_1691del TGGCCCAA p.(Pro563Hisfs*14) | 1 | 869391 in ClinVar | Chr19: 11226866-11226875 | - | EGF spacer | Pathogenic (PVS1 PM1 PM2 PP3) | D | D | -- | - |
B, Genetic variants in |
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8 | Missense Exon 2 c.100T>G p.(Cys34Gly) | 1 | rs879254405 | Chr19: 11210931 | - | Ligand-binding | Pathogenic/ Likely pathogenic | D | D | D | ( |
9 | Frameshift Exon 4 c.316_328delCCC AAGACGTGCT p.(Lys107Argfs*95) | 1 | LDLR_001035 in LOVD database | Chr19: 11215901-11215915 | - | Ligand-binding | Pathogenic | D | D | -- | ( |
10 | Missense Exon 4 c.552T>G p.(Cys184Trp) | 1 | LDLR_000858 in LOVD database | Chr19: 11216134 | - | Ligand-binding | Likely pathogenic | D | D | D | ( |
11 | Missense Exon 5 c.798T>A p.(Asp266Glu) |
1 | rs139043155 | Chr19: 11217344 | 0.000032 | Ligand-binding | Pathogenic/ Likely pathogenic | D | D | - | ( |
12 | Missense Exon 6 c.887G>A p.(Cys296Tyr) | 1 | rs879254707 | Chr19: 11218137 | - | Ligand-binding | Likely pathogenic | D | D | D | ( |
13 | Nonsense Exon 6 c.888C>A p.(Cys296*) | 1 | rs879254708 | Chr19: 11218138 | - | Ligand-binding | Pathogenic | D | D | -- | ( |
14 | Missense Exon 6 c.938 G>A p.(Cys313Tyr) | 1 | rs875989910 | Chr19: 11218188 | - | Ligand-binding | Pathogenic/ Likely pathogenic | D | D | D | ( |
15 | Missense Exon 7 c.986G>A p.(Cys329Tyr) |
2 | rs761954844 | Chr19: 11221373 | 0.000016 | EGF precursor homology repeat A | Likely pathogenic | D | D | D | ( |
16 | Nonsense Exon 7 c.1048C>T p.(Arg350*) | 1 | rs769737896 | Chr19: 11221435 | - | EGF precursor homology repeat A | Pathogenic | D | D | -- | ( |
17 | c.1186+1G>T | 1 | rs730880131 | Chr19: 11222316 | - | Splice donor site‡, intron 8 | Pathogenic/ Likely pathogenic | -- | D | -- | - |
18 | Missense Exon 9 c.1202T>A p.(Leu401His) | 3 | rs121908038 | Chr19: 11223969 | - | EGF spacer | Likely pathogenic | D | D | D | ( |
19 | Missense Exon 9 c.1277 T>C p.(Leu426Pro) | 1 | rs879254851 | Chr19: 11224044 | - | EGF spacer | Pathogenic/ conflicting- interpretations-of- pathogenicity | D | D | B | ( |
20 | Frameshift Exon 10 c.1478_1479delCT p.(Ser493Cysfs*42) | 1 | rs869025453 | Chr19: 11113652-11113655 | 0.00003 | EGF spacer | Pathogenic/ Likely pathogenic | -- | D | -- | ( |
21 | Missense Exon 12 c.1730G>C p.(Trp577Ser) | 1 | rs138947766 | Chr19: 11227559 | 0.000008 | EGF spacer | Pathogenic/ Likely pathogenic | D | D | D | ( |
22 | Missense Exon 12 c.1775G>A p.(Gly592Glu) | 6 | rs137929307 | Chr19: 11227604 | 0.000044 | EGF spacer | Pathogenic/ Likely pathogenic | D | D | D | ( |
23 | Nonsense Exon 15 c.2230C>T p.(Arg744*) | 1 | rs200793488 | Chr19: 11233939 | 0.000004 | O-linked sugars | Pathogenic | D | D | - | ( |
C, Genetic variants in |
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24 | Missense Exon 26 c.9175C>T p.(Arg3059Cys) |
1 | rs146377316 | Chr2: 21230565 | 0.000008 | LDLR binding | Unknown significance | B | B | B | ( |
25 | Missense Exon 26 c.10580G>A p.(Arg3527Gln) |
1 | rs5742904 | Chr2: 21229160 | 0.000275 | LDLR binding | Pathogenic | B | D | D | ( |
26 | Missense Exon 26 c.10580G>T p.(Arg3527Leu) | 1 | rs5742904 | Chr2: 21229160 | - | LDLR binding | Pathogenic | D | D | D | ( |
27 | In-frame deletion Exon 29 c.13480_ 13482delCAG p.(Gln4494del) |
2 | rs562574661 | Chr2: 21001940-21001945 | 0.000384 | - | Likely pathogenic/ conflicting- interpretations-of- pathogenicity | - | B | - | ( |
D, Genetic variants in |
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28 | Nonsense |
1 | rs199689137 | Chr2: 44050063 | 0.00018 | Cytoplasmic | Pathogenic | D | D | - | ( |
29 | Missense |
1 | rs137852987 | Chr2: 44099233 | 0.00102 | Cytoplasmic | Pathogenic | D | D | - | ( |
30 | Missense |
1 | rs201690654 | Chr2, 44102425 | 0.000215 | Transmembrane | Unknown significance | D | D | D | ( |
E, Genetic variant in |
|||||||||||
31 | c.894G>A p. (Q298=) | 1 | rs116928232 | Chr10: 89222511 | 0.00083 | Exon skipping mutation | Pathogenic | -- | D | -- | ( |
F, Genetic variant in |
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32 | Missense Exon 9 c.1486C>T p.(Arg496Trp) | 1 | rs374603772 | Chr1, 55524303 | 0.000044 | LDLR-binding | Unknown significance/ conflicting- interpretations-of- pathogenicity | D | D | D | ( |
aThe frequency of the identified variants was additionally assessed following in-house exome databases. Novel variants were not found in the Russian 870 exomes and the Northwest Russia 694 exomes databases.
bThe prediction of the pathogenicity was performed using SIFT, PolyPhen-2 and MutationTaster tools if suitable. For two intronic variants, analysis with Human Splicing Finder was performed: These variants alter canonical splice donor sites in introns 6 and 8 of the
cPathogenicity prediction for novel variants was performed according to American College of Medical Genetics and Genomics-based classification, for already characterized variants-classification according to ClinVar database.
dThese variants were functionally characterized by